Band 39, Heft 12

Viral hepatitis is one of the most prevalent infectious diseases in China. To date, all five types, hepatitis A, B, C, D and E have been reported in China, and the incidences of all these types are high in the Chinese population. Serological tests are mainly used for the diagnosis of hepatitis A, B, C and E in patients, epidemiological surveys and for efficacy studies of vaccines. Currently, nucleic acid-based assays are only used in research and for evaluation of antiviral and immuno-therapies.

Molecular diagnostics is changing the face of clinical laboratories and laboratory medicine. The case of China is no exception. In the present paper, a brief description on this promising discipline is given first, followed by an overview of the development of molecular diagnostics in China. Work done in the author's own laboratory is introduced in the third part and a short discussion on the challenges ahead is provided last.

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical AlDH 2 2 allele have higher levels of acetaldehyde in their plasma. The atypical AlDH 2 2 allele has a nucleotide base transition (G→A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual AlDH 2 1 allele and the atypical AlDH 2 2 allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical AlDH 2 2 allele was found to be 12%.

Lipocalin-type prostaglandin D synthase (L-PGDS) has recently been shown to be expressed in human brain tumors and breast tumors. However, L-PGDS expression has not been investigated in ovarian cancer. The objective of this study was to determine whether L-PGDS is expressed in human ovarian cancer. Lipocalin prostaglandin D synthase mRNA was cloned and sequenced by RT-PCR. Using in situ hybridization (ISH) technique, the expression of L-PGDS mRNA in 54 ovarian cancer was investigated. Expression of L-PGDS mRNA was found in tumor cells of all various types of ovarian cancers. Patterns of staining of tumor cells varied among different histological types of ovarian cancer. Significant discrepancy between the intensity of the staining and histological types of ovarian cancer could be established (p<0.01). It is reported for the first time that the expression of mRNA of L-PGDS exists in the ovarian cancer, and is related to the cancer type. This may have significance for the progress of ovarian cancer.

The aim of this study was to establish a simple, rapid carrier detection and prenatal diagnosis system for hemophilia A. Intron 22 inversion in FVIII gene was directly examined by long-distance polymerase chain reaction. Polymorphisms of factor VIII intragenic restriction fragment length polymorphism of Bcl I , short tandem repeat (STR) within intron 13 and 22, and extragenic DXS 52 (St 14) variable number tandem repeats (VNTR) loci were assessed by hereditary linkage analysis. The diagnostic rates for these loci were 47.6% (intron 22 inversion), 27.8% ( Bcl I ), 28.6% and 29.4% ( STR within intron 13 and 22), and 81.3% ( DXS52 ), respectively. The overall diagnostic rate in 21 families was 94.7%. The diagnosis in hemophilia A patients or carriers can be made if intron 22 inversion is present. The intragenic and extragenic loci hereditary linkage analysis could be used to establish the diagnosis in intron 22 inversion-negative patients.

Many disorders of the central nervous system are associated with increased concentration of cerebrospinal fluid protein due to either an increase in the permeability of blood-brain barrier or the synthesis of immunoglobulins within the central nervous system. Immunofixation electrophoresis was used to detect the immunoglobulins in sera and cerebrospinal fluid in patients with different central nervous system disorders. A strong oligoclonal band, or a single weak monoclonal band, observed in the cerebrospinal fluid but not in the serum, indicates the endogenous immunoglobulin synthesis. In 17 cases of non-infectious central nervous system diseases, we diagnosed one case of progressive multifocal leukoencephalopathy, one case of multiple sclerosis , one case of Guillain-Barre syndrome, and one case of epilepsy. A weak band was observed in both serum and cerebrospinal fluid samples in two cases of cerebral trauma. Out of 25 cases of infectious central nervous system diseases, two cases of nervous system syphilis were positive only in cerebrospinal fluid samples, and 23 cases were negative in both samples. Compared with the standard immunofixation, the application of an enzyme-labeled antibody can significantly increase the sensitivity of the method.

During the 2nd Sino-European Congress in Shanghai in April 2001 the aim of the presentation was an overview on strategies and systems of laboratory diagnostic work. The main focus was to define the prerequisites and the different approaches in the diagnostic work in patients' treatment. In this paper, besides the area of emergency medicine with definition of vital functions and the routine work in deciding between healthy and ill individuals and in the control of therapy, the different aspects in the field of research and teaching are specified. Some aspects of workflow definition and optimization are discussed.

Aim: This study is aimed to discuss the present situation, existing problems, and relevant measures concerning the management of clinical laboratories in China. Methods: This study was carried out through the analysis of the problems relevant to the quality management of the laboratory medicine, based on the advanced experiences of ISO and developed countries in the management of clinical laboratories. Results: Many problems still remain unsolved concerning the quality management of clinical laboratories. Conclusion: Only legalized and standardized management guarantees the quality of clinical inspection.

A software has been developed on quality control, department economics, information supervise and personal administration. In this article, we discuss the part of quality control, from which we benefit most, on developing ideas, running principle, and application. This network will enable the implementation of total quality control, and possibly total computerised management.

Synovial fluid fills the spaces in the joint cavities. Many diseases can affect the joints and frequently only a direct examination of the tissue or synovial fluid will yield the correct diagnosis. The article presents the basis of the synovial fluid analysis and a relevant decision- making scheme. The reliability and applicability of synovial fluid tests are commented upon. The synovial fluid analysis undoubtedly plays an important role in the management of patients with joint diseases. Due to a lack of interlaboratory comparisons, and therefore expert opinions on the interpretation of results, this knowledge and the acquisition of relevant experience should be encouraged to enable evaluation of the clinical applicability of recent discoveries in synovial fluid pathology.

The determination of tumor markers may have consequences for the patients' treatment, which requires special attention to the analysis and to the expression of the results. In addition to the factors usually dealt with in the pre-analytical phase (identification, quality and storage of the sample) and in the analytical phase (interference, endogenous antibodies, hook effect), we must consider factors such as normal values (which depend heavily on the used techniques), threshold values defining other characteristics (sensitivity, specificity, positive and negative predictive value) and kinetics. Knowledge of the limitations of the tumor marker analysis, in particular tumor markers for non-cancerous diseases leading to possible increases (false positives), is also indispensable. All of this led to the definition of guidelines for good practice, including the design of quality control, rules for requesting, validation and interpretation of results, and the setting up of a serum library.

The urinary 6β-OH-cortisol/cortisol ratio is a specific, non-invasive marker for evaluating inductive or inhibitory effects on cytochrome P450 3A activity. We propose a new quantitative gas chromatographymass spectrometry with isotope dilution (GC-ID-MS) method for the simultaneous determination of urinary free cortisol (UFC) and 6β-OH-cortisol (6β-OHC). The method utilizes the following: (a) addition of internal standard ( 2 H 2 cortisol) to 1 ml of urine; (b) loading on to an Extrelut column and elution with dichloromethane; (c) derivatization to dimethoxime tri-(trimethyl-silyl)-ether (MOX-TMS); (d) separation and identification by GC-ID-MS. The detection limit for cortisol was 22 pg injected (signal-to-noise ratio 10:1) and for 6β-OH-cortisol 123 pg injected (signal-to-noise ratio 10:1). The intra-assay and the inter-assay imprecision were 4.69% and 7.4% for 6β-OHC and 2.44% and 3.53% for cortisol, respectively. We used this method to analyze 57 morning urine samples of healthy volunteers and patients under different conditions. We found that chronic alcoholics had a significantly higher ratio of 6β-OHC/UFC compared to controls (p<0.0001), whereas adults undergoing methadone therapy and patients with acute alcohol intoxication exhibited a significantly lower urinary 6β-OHC/UFC ratio (p<0.05 and p<0.01, respectively). The proposed method allows a rapid and accurate assessment of the 6β-OHC/UFC ratio.

We evaluated mRNA expression of the heat shock protein gene, Hsp70-1 , by means of a semiquantitative RTPCR in atrial tissue specimens from pediatric patients collected before and after cardiopulmonary bypass surgery for congenital heart diseases, to see whether surgical stress may affect the expression level of this mRNA. We studied thirty nine pediatric patients (aged 3 months to 15 years) undergoing surgical correction of congenital heart malformation. Twenty-one patients were affected by the tetralogy of Fallot, two by combined atrioventricular septal defects, six by ventricular septal defect, three by atrial septal defect, two by atrioventricular canal defect, two by pulmonary valve stenosis, one by mitral insufficiency, and one by double-outlet right ventricle. Our results showed no significant changes in the Hsp70-1 mRNA expression in atrial tissue of patients before and after cross-clamping (the mean relative expression after cross-clamping was 1.0±0.6 compared to the baseline value). Furthermore, no significant correlations were observed between cross-clamping time and temperature, cardiopulmonary bypass time and mRNA variation. Our study suggests that, during cardioplegia, myocardial tissue does not have an appropriate adaptive response to surgical stress.

Immunoprecipitation with anti-human immunoglobulin G-agarose was evaluated for the detection of prolactin-immunoglobulin G (PRL-IgG) complexes in macroprolactinemic samples from hyperprolactinemic patients (prolactin measured using the automated Elecsys PRL assay; Roche Diagnostics, Mannheim, Germany). Using the polyethylene glycol (PEG) precipitation test on serum samples with PRL above 1000 mIU/l, we detected macroprolactin in 38 out of 175 samples with a recovery below 50%. Gel filtration chromatography on a subset of hyperprolactinemic samples confirmed that macroprolactin was the predominant immunoreactive form of PRL in samples which showed a recovery ≤50%. In 37 out of 38 samples containing macroprolactin, immunoprecipitation with anti-human IgG (anti-hIgG)-agarose revealed a higher PRL-binding (19.1–91.3%) than in the sera containing monomeric PRL (<10%). In conclusion, we found that PEG precipitation detected macroprolactin in 21.7% of samples with elevated PRL levels as measured by the Elecsys PRL assay, and that in the vast majority of these samples the presence of PRL-IgG complexes could be demonstrated by immunoprecipitation with anti-hIgG-agarose. In view of the high prevalence of PRL-IgG complexes in hyperprolactinemic patients and the simplicity of the test, immunoprecipitation with anti-hIgG-agarose might play a role in the routine laboratory handling of hyperprolactinemic samples, especially with regards to PRL assays where PEG causes interference.

The haploinsufficiency of chromosome 22q11.2 can cause both DiGeorge and velocardiofacial syndromes, both of which are characterized by conotruncal heart defects as well as a wide range of other extracardiac anomalies. Several studies have demonstrated that approximately 10–20% of patients with conotruncal heart defects have a 22q11.2 deletion. In clinical laboratories, the deletion is usually detected by fluorescent in situ hybridization (FISH). We set up a polymerase chain reaction-based non-radioactive method for molecular analysis of the 22q11.2 region in conotruncal cardiac patients with conotruncal defects. Sixty-four children with conotruncal defects and their parents were genotyped by polymerase chain reaction, using fifteen polymorphic markers. We identified nine deletions (confirmed by FISH): eight were “de novo” and one familial, maternally inherited. Six deletions were of paternal and three of maternal origin. There were seven deletions of 3 Mb and the other two were of 1.5 Mb. This method is a cost-effective means of characterizing the 22q11.2 region and it can be applied for a rapid screening of 22q11.2 deletion in patients at risk. In agreement with previously published data, we found no correlation between the sizes and the parental origin of deletions and cardiac or extra-cardiac phenotypes.

It is now known that 15 monogenic, mostly neurological, disorders are caused by the same type of mutations that occur in trinucleotide repeat sequences in certain genes. Since they share a nonspecific and variable phenotype, the accurate diagnosis could be made only by DNA analysis. We developed an Expand Long PCR assay that provides more reliable molecular diagnosis of such disorders. Its main characteristics are robust amplification of expanded alleles, simplicity, low cost and speed. We suggest the use of Expand Long PCR for routine molecular diagnosis of triplet repeat diseases, and present such analysis of the fragile X syndrome, myotonic dystrophy and Huntington's disease.

Endotoxin-induced peroxynitrite formation has been demonstrated in plasma. The aim of this study is to evaluate whether this has an effect on erythrocytes. For this purpose erythrocyte 3-nitrotyrosine (3-NT) level, Na + -K + ATPase and glutathione peroxidase activities were measured both in vivo and in vitro . In vivo peroxynitrite formation was induced in rats by intraperitoneal Escherichia coli ( E.coli ) injection. Erythrocytes were directly incubated with peroxynitrite in the in vitro experiment. 3-NT levels were measured by reverse-phase HPLC, glutathione peroxidase, and Na + -K + ATPase activities were measured by spectrophotometric techniques. There was a marked increase in the 3-NT levels in both experiments. However, glutathione peroxidase activity was significantly increased in in vivo experiments, while decreasing in in vitro conditions. Although Na + -K + ATPase activities were significantly reduced by peroxynitrite in vitro , Na + -K + ATPase activities were similar in control and E.coli -injected rat erythrocytes. Although nitrating effect of peroxynitrite does not seem to be preventable by endogenous antioxidants, this effect of peroxynitrite may not endanger erythrocytes if the oxidative damage of peroxynitrite is prevented.

Undesirable interactions between anticoagulants and diagnostic test kit procedures so far have prevented the development of a single uniform blood sampling tube. Contrary to K 2 -EDTA, heparin and other anticoagulants, hirudin only minimally alters blood cells and dissolved blood constituents, thus qualifying as a universal anticoagulant for diagnostic purposes. Automated complete blood counts, automated analyses of clinical chemistry analytes and immunohaematology were performed from hirudinised and routinely processed blood obtained from healthy volunteers (n=35) and hospitalised patients (n=45). Hirudin (400 ATU/ml blood) sufficiently anticoagulated blood for diagnostic purposes. The measurements of automated complete blood counts obtained from K 2 -EDTA-anticoagulated and hirudinised blood correlated significantly as did the measurements of 24 clinical chemistry analytes from hirudinised plasma and serum. Regression analysis revealed that the results of complete blood counts and clinical chemistry tests were predictable from the respective measurements from hirudinised blood (p=0.001). Immunohaematological tests and crossmatching from hirudinised and native blood of the same donors gave identical results. Single clotting factors, but not global coagulation analytes, could be measured from hirudinised blood. Therefore, a universal hirudin-containing blood sampling tube could be designed for automated analysis of haematological, serological and clinical chemistry analytes.

We compared six routinely employed immunoassay kits: Architect i2000 and AxSYM, Abbott Laboratories; Elecsys 2010, Roche Diagnostics; ELSA, CIS-BioInternational; Immulite 1, Diagnostic Products Corporation; and IRMA-mat, Byk-Sangtec Diagnostica. Using all analytical systems, we measured identical groups of clinical samples completed with selected control samples. The repeatability of measurements (coefficient of variation) ranged from 2.1% (Elecsys 2010) to 6.7% (ELSA). The parameters of Passing-Bablok regression show significant systematic differences among analytical systems. Data from a Bland-Altman diagram suggest that these differences project onto other, still more significant individual differences among individual samples. Though the cut-off values differ between various systems, no similar clinical efficacy appears to be attained. The behavior of individual systems is quite different for identical control materials and does not necessarily duplicate the calibration for biological samples. The results of determining CA 19–9 cannot be extrapolated from one analytical technique to another, even in cases where the same monoclonal antibody is used. Standardization of CA 19–9 measurement systems is necessary to allow use of the results for the purposes of evidence-based medicine.

In this study analytical and functional performance of the Precision G TM “point-of-care” glucometer (MediSense Inc.) was evaluated. Studies were carried-out using capillary blood collected for routine monitoring of glycemia in diabetic patients. Each glucose test measurement with the glucometer was paralleled by the laboratory measurement of glucose on the same blood sample, using the GOD/PAP method. Mean accuracy error in the glucose concentration range of 1.1–33.3 mmol/l calculated for the glucometer vs. the laboratory method amounted to only 0.2%. However, for glucose concentrations below 4.4 mmol/l the mean accuracy error was 3.9%, and for the concentrations above 10.0 mmol/l it was 4.6%. Within-run CV for three concentration levels was 2.76%, 2.89%, and 4.22%, respectively. Linearity of the meter response in samples with glucose concentration ranging from 1.7 mmol/l to 16.7 mmol/l, expressed as the correlation coefficient r, yielded r=0.996 and linear regression equation [y 1 = 0.996 y 2 –0.005], where y 1 is the measured glucose concentration and y2 is the target glucose concentration calculated in diluted samples. Correlation studies on a set of 114 blood samples collected from patients and assayed by glucometer and by the laboratory method yielded a relationship expressed by the equation: y = 0.84x + 1.13 where y is glucometer read-out and x is glucose concentration obtained by the laboratory method. Passing-Bablok test showed a significant agreement between the glucometer measurements and the reference laboratory results in the studied glucose concentration range. The error grid analysis of series of the paired patient's samples showed that 95% of results were in the clinically acceptable zone A and 1% of results in zone

This article considers the 18 century European Enlightenment as an example of the cross-fertilization between philosophy propounding rationalist-empirical approach, and arts. The example of Sir Henry Raeburn, a Scottish portraitist, is given to illustrate the influence of the concepts of perception developed by Thomas Reid, the founder of the Scottish school of common sense philosophy, on the style of painting.